Analysis Of Cut-wounding Transcriptome And PPOs Gene Expression In Stem-ends Of Cut Lily Flowers

Cut flowers are mainstream ornamental products in the domestic and international flower markets, their stems are severely wounded. Previous researches showed that phenolic oxidation metabolism in wounds and the next parts would be significantly induced once plant tissueswere wounded. However, so far there are few researches on understanding the wounding response of cut flowers and its molecular mechanism. Thus, this thesis firstly cloned and characterized the genes of polyphenol oxidase(PPO) in cut Lilium Oriental-Trumpet hybrid ‘Robina’, which is a key enzyme in phenolic oxidative metabolism, and then focused on the transcriptome of cut-wounding in stem-ends of cut Lilium Oriental hybrid ‘Tiber’. The major research results were as follows:(1) Three PPOs genes, LhPPO1, LhPPO2 and LhPPO3, were cloned from cut lilies ‘Robina’, their cDNA sequences contain 1 668, 1 683 and 1 692 bp long opening reading frame(ORF), and encoding a protein of 555, 560 and 563 amino acids, respectively. All predicted PPO proteins contained typical conserved domains, including tyrosinase superfamily structure motif, PPO1_DWL motif and PPO1_KFDV motif. The promoter sequences of LhPPO1 and LhPPO2 were obtained by Tail-PCR, they were 1 888 bp and 1 095 bp long, respectively. It was found that there were a series of cis-acting elements responsive to wounding, pathogens and plant hormones besides the known basic cis-acting elements of promoters. LhPPO1 and LhPPO2 were highly expressed in stamens and stem of cut lily flowers, respectively. However, the expression of LhPPO3 was not observed in pistils, stamens, petals, leaves and stems.(2) There were 7 805 differentially expressed genes(DEGs) in the result of RNA-seq of cut-wounding in cut lilies ‘Tiber’. In general, in the early period(within 6 h) of the wound response, there is a lot of unigenes showed a trend of up-regulated expression, however, thereafter the number of down-regulated genes gradually increased. Based on a comparison with the KEGG database, the most major pathways were ‘plant hormone signal transduction’(73 DEGs), ‘plant-pathogen interaction’(53 DEGs), ‘starch and sucrose metabolism’(46 DEGs), ‘phenylpropanoid biosynthesis’(34 DEGs), ‘protein processing in endoplasmic reticulum’(32 DEGs) and ‘phenylalanine metabolism’(28 DEGs). Further analysis indicated that ‘plant hormone signal transduction’, ‘plant-pathogen interaction’ and ‘phenylpropanoid biosynthesis’ were the top three pathways in regards with the reliability and enrichment degree, which implied that these pathways may play very important roles in plant wound response.(3) The 3 PPOs genes from cut lilies ‘Robina’ were much similar to corresponding sequence from transcriptome sequencing of cut lilies ‘Tiber’, with similarity of 82.2%, 97.8%, 94.5% respectively, and their expression trend was also very similar. Additionally, the expression of PPOs genes in there two varieties of cut lilies both showed a quick and significant up-regulation within 6 h after cutting, and the high level expression of LhPPO2 and c62702.graph_c0 could maintain a relatively long time, i.e. from 6 h to 48 h and 168 h after cutting, respectively.